Amphotericin B (AMB) is an effective antifungal agent, and at present, is the drug of choice for most serious fungal infections (reference 1). The drug binds strongly to ergosterol, a major sterol component of fungal membranes, forming pores in the membranes which allow leakage of solute molecules.
The drug also has a strong binding affinity for cholesterol, a sterol present in most mammalian cell membranes, and is therefore capable of disrupting host cells. When AMB is administered in free form, side effects resulting from red blood cell disruption are observed initially, followed by more serious cardiotoxicity effects. Renal toxicity, resulting from the body's attempt to clear the drug, is also present.
Several studies have shown that AMB toxicity can be reduced significantly by administering the drug in a liposome-bound form (references 2-12). Typically, the LD50 of the drug increases from about 2-3 mg/kg body weight for the free drug up to about 8-15 mg/kg when the drug is administered in liposomal form. The decreased toxicity of liposome-associated AMB is presumably related to the ability of the liposomal membrane, and particularly sterol components in the membrane, to compete with host cell membrane for binding to the drug. Ergosterol, which has a higher affinity for AMB than cholesterol, shows a greater protective effect than cholesterol in liposomes (reference 12). However, cholesterol-containing liposomes allow more favorable AMB exchange between liposomes and the fungal target membranes, and are thus generally more beneficial as a carrier for therapeutic AMB.
For a variety of reasons, it is desirable that AMB liposomes which are used for therapeutic purposes have stable, sizes in a defined size range less than about 1 micron. Cholesterol-containing liposomes with sizes greater than about 1-2 microns are generally more toxic than smaller liposomes when administered parenterally, i.e., into the bloodstream. The toxicity of large liposomes in the bloodstream is related in part to liposome blockage of the alveolar capillaries. There are also indications that larger liposomes are more toxic to the liver, presumably due to accumulation of large liposomes in reticuloendothelial cells.
In addition to toxicity effects, liposome size may be an important determinant for in vivo drug release rates and liposomes targeting. For example, where AMB liposomes are injected intramuscularly, the size of the liposomes may be an important variable in controlling either liposome migration and/or drug release from the site of injection. Liposomes have also been used in administering drugs by inhalation, as described in co-owned patent application for "Liposome Inhalation System and Method", Ser. No. 737,221, filed May 22, 1985. In this system, control of liposome sizes may be important in achieving reproducible drug delivery and/or drug release characteristics.
Heretofore, attempts to produce AMB liposomes having stable, selected sizes have not been successful. This is because AMB, like other polyene antibiotics, destabilizes liposome membranes, causing liposome fusion and general size increase over time For example, AMB liposomes which have an initial average size less than about 1 micron typically show a 200-300% increase in average size after one month of storage at refrigerator temperature.